title: Peptide transporter DtpA has two alternate conformations, one of which is promoted by inhibitor binding.
abstract: Peptide transporters (PTRs) of the large PTR family facilitate the uptake of di- and tripeptides to provide cells with amino acids for protein synthesis and for metabolic intermediates. Although several PTRs have been structurally and functionally characterized, how drugs modulate peptide transport remains unclear. To obtain insight into this mechanism, we characterize inhibitor binding to the Escherichia coli PTR dipeptide and tripeptide permease A (DtpA), which shows substrate specificities similar to its human homolog hPEPT1. After demonstrating that Lys[Z-NO2]-Val, the strongest inhibitor of hPEPT1, also acts as a high-affinity inhibitor for DtpA, we used single-molecule force spectroscopy to localize the structural segments stabilizing the peptide transporter and investigated which of these structural segments change stability upon inhibitor binding. This characterization was done with DtpA embedded in the lipid membrane and exposed to physiologically relevant conditions. In the unbound state, DtpA adopts two main alternate conformations in which transmembrane α-helix (TMH) 2 is either stabilized (in ∼43% of DtpA molecules) or not (in ∼57% of DtpA molecules). The two conformations are understood to represent the inward- and outward-facing conformational states of the transporter. With increasing inhibitor concentration, the conformation characterized by a stabilized TMH 2 becomes increasingly prevalent, reaching ∼92% at saturation. Our measurements further suggest that Lys[Z-NO2]-Val interacts with discrete residues in TMH 2 that are important for ligand binding and substrate affinity. These interactions in turn stabilize TMH 2, thereby promoting the inhibited conformation of DtpA.
authors.affiliation: Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule Zürich, 4058 Basel, Switzerland.
authors.forename: Christian A
authors.initials: CA
authors.lastname: Bippes
authors.forename: Lin
authors.initials: L
authors.lastname: Ge
authors.forename: Marcel
authors.initials: M
authors.lastname: Meury
authors.forename: Daniel
authors.initials: D
authors.lastname: Harder
authors.forename: Zöhre
authors.initials: Z
authors.lastname: Ucurum
authors.forename: Hannelore
authors.initials: H
authors.lastname: Daniel
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Fotiadis
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Müller
mesh_terms: D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics; D009842:Oligopeptides / Q000737:chemistry*; D000074011:Peptide Transporter 1; D055550:Protein Stability; D017433:Protein Structure, Secondary; D021381:Protein Transport / Q000502:physiology; D040681:Structural Homology, Protein; D027981:Symporters / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D009842:Oligopeptides; D000074011:Peptide Transporter 1; C082186:SLC15A1 protein, human; D027981:Symporters; C037230:oligopeptide permease, Bacteria
keywords: atomic force microscopy; major facilitator superfamily; membrane transporter; molecular interactions; proton-dependent oligopeptide transporter